Coffee is an agricultural commodity that plays a significant role in the economies of many developing countries. In Colombia, coffee cultivation is restricted to mountain areas with altitudes between 1200 and 1400 meters above sea level. It is especially concentrated in the central region, in an area called the Coffee Zone. With a total annual yield of around 12 million bags, Colombia is ranked second in world production. Of this production, 26% is used for domestic consumption, and the rest is exported to Europe (6 million bags), United States (3 million bags), and Asia (1 million bags), with an average annual market value (1991 to 1995) of $1.6 billion (Banco de la Republica, Indicadores Economicos NI 828 Banco de la Republica, Bogota, Colombia (1996)).
The genus Coffea belongs to the Rubiaceae family which includes other important plants, such as ipecacuanha (Cephaelis ipecacuanha) and cinchona (Cinchona spp.). The genus contains about 70 species, most of them trees and shrubs growing at low altitudes in the tropical rain forests of Africa and Asia (Sondahl et al., “Coffee,” Biotechnology of Perennial Crops CAB International, Wallingford, UK (1992)). Only two species are widely cultivated, Coffea arabica and Coffea canephora. All known species are diploid (2n=2X=22 chromosomes) and obligate outbreeders with self-incompatibility systems, except for C. arabica which is tetraploid (2n=4X) and self-fertile.
The species Coffea arabica L probably originates from a relatively recent cross between C. eugenoides and C. canephora, a hypothesis supported by random amplified polymorphic DNA's (RAPD) (Lashermes et al., “Use of Amplified DNA Markers to Analyze Genetic Variability and Relationships of Coffea Species,” Genetic Resources and Crop Evolution 40:91-99 (1993)) and chloroplast restriction fragment length polymorphism (RFLP) analyses (Lashermes et al., “Inheritance and Restriction Fragment Length Polymorphism of Chloroplast DNA in the Genus Coffea L.,” Theoretical and Applied Genetics 93:626-632 (1996)). The nuclear DNA content of C. arabica, as determined by flow cytometry, is 2.4 pg/interphase nucleus, or n=2X=1158 Mb (Arumuganathan et al., “Nuclear DNA Content of Some Important Plant Species,” Plant Molecular Biology Reporter 9:208-218 (1991)). It is cultivated in 75% of the coffee plantations around the world. The quality of the beverage is potentially excellent, being known in the trade as “mild coffee.” The most important pests affecting this species are coffee rust (Hemileia vastatrix), coffee berry disease (CBD, Colletotrichum coffeanum), and coffee berry borer (Hypothenemus hampei, Coleoptera). Worldwide, these three pests cause an estimated crop loss of 14.8%, or about $1 billion annually (Oerke et al., “Estimated Losses in Major Food and Cash Crops,” Crop Production and Crop Protection, Elsevier, N.Y., U.S.A. (1994)).
Several cultivars have been described for C. arabica, but because of the narrow genetic base of the species, they are due mainly to single gene mutations. The commonly grown varieties, Tipica and Bourbon, can grow up to 6 m tall under natural conditions. Coffee trees grow well at tropical elevations, ranging from 300 to 1200 m above sea level, with a mean annual temperature of 18 to 21° C. C. arabica cv. Caturra is a mutant of the Bourbon cultivar, which was discovered in Brazil in 1949 and has been extensively grown in Colombia. The main characteristic of this cultivar is the dwarf phenotype resulting from the action of a dominant gene that reduces the internode distance (Orozco, “Descripcion de Especies y Variedades de Café,” CENICAFE Chinchiná, Caldas (1986)). Use of this phenotype has allowed planting densities to increase from 2500 plants to 10,000 plants/h, which, in turn, has increased bean yields from 5000 kg to about 8000 kg/h.
The species Coffea canephora Pierre ex Froehner, also known as Coffea robusta Linden, is the diploid species most widely cultivated around the world. It is self sterile and cross pollinated and therefore much more variable than C. arabica. C. canephora is better adapted to humid-hot climates and is frequently cultivated in low to medium altitudes. The quality of the beverage made from C. canephora is usually regarded as inferior to that made of C. arabica. However, C. canephora is more resistant to coffee rust and CBD.
Traditionally, Tipica, Bourbon, and Caturra were the C. arabica cultivars grown in Colombia. These varieties produce a high quality coffee, but they are very susceptible to pests which are not held in balance by natural biocontrol. Although South America was free of the most important coffee pests for many years, threats became real with the appearance of coffee rust in Brazil in 1970 and in Nicaragua in 1976. This disease finally arrived in Colombia in 1983. In anticipation, the Colombian National Center of Coffee Research (CENICAFE), a organization of Colombian coffee growers, began a breeding program for resistance to coffee rust in 1968. The purpose was to create a cultivar of C. arabica that preserves the traditional cup quality, but incorporates increased genetic diversity, durable resistance to the coffee rust, phenotypic homogeneity, and productivity (Castillo et al., “La Variedad Colombia: Selección de un Cultivar Compuesto Resistente a la Roya del Cafeto,” CENICAFE Chinchiná, Colombia, 171 p. (1986)).
Timor hybrid was chosen as the resistant parent of the new cultivar since no germplasm of C. arabica was known to contain durable resistance genes against coffee rust. Timor hybrid is a natural interspecific hybrid between C. arabica and C. canephora found in 1917 on the island of Timor, Indonesia. Used in Africa and India for many years, it showed broad resistance against the local rust races. In the Colombian breeding program, the recurrent quality parent of the new cultivar was C. arabica cv. Caturra, which in addition to providing the characteristics of dwarfism and good beverage quality, was a familiar cultivar among the growers. The result was the release in 1980 of the Colombia cultivar, a composite cultivar made up by the mixture of seeds coming from the best F5 and F6 progenies resistant to coffee rust and with optimal adaptation to the climate and soils of the Colombian coffee zone (Castillo et al., “La Variedad Colombia: Selección de un Cultivar Compuesto Resistente a la Roya del Cafeto,” CENICAFE Chinchiná, Colombia, 171 p. (1986)).
Components of the Colombia cultivar are continuously tested for their resistance against coffee rust and other diseases. When a component is found susceptible, it is withdrawn from the mixture. In the same way, new selected components can be added to the cultivar. This procedure provides a dynamic update of the cultivar in its resistance against coffee rust. Seed production and distribution of the Colombia cultivar are carried out exclusively by the National Federation of Coffee Growers. This maintains a diversity in resistance to coffee rust as well as the phenotypic homogeneity, yet results in low seed prices for the farmers.
In contrast to many other crops, coffee has not been the subject of extensive research in molecular biology. This may be due to factors such as the long life cycle, the difficulty of maintaining plants out of the tropical environment, and the lack of resources from countries that cultivate coffee. Nevertheless, some advances are being made in this field.
Several proteins, especially those involved in the resistant interaction with coffee rust have been studied. Kinetics and differential expression of phenylalanine ammonia lyase (PAL) (Almario, “Study of the Activity of the Phenylalanine Ammonia Lyase in the Presence of the Pathogen in Coffee Varieties Resistant and Susceptible to Hemileia vastatrixBer & Br.,” Universidad Nacional de Colombia, Bogota, 155 p. (1992)), superoxide dismutase (Daza et al., “Isoenzyme Pattern of Superoxide Dismutase in Coffee Leaves from Cultivars Susceptible and Resistant to the Rust Hemileia-Vastatrix,” Journal of Plant Physiology 141:521-526 (1993)) and lipoxygenase (Rojas et al., “Stimulation of Lipoxygenase Activity by Cotyledonary Leaves of Coffee Reacting Hypersensitively to the Coffee Leaf Rust,” Physiological and Molecular Plant Pathology 43:209-219 (1993)) of the Caturra and Colombia cultivars have been compared.
Phenylalanine ammonia lyase (PAL) is a key enzyme that catalyzes the deamination of L-phenylalanine to produce cinnamic acid. Cinnamic acid is a substrate that feeds several biosynthetic routes, leading to the production of various classes of phenylpropanoid-derived secondary plant products. Some of these products are involved in aspects of the normal development of the plant such as petal pigmentation and xylem development. However, many of them are directly involved in the plant defense response (Hahlbrock et al., “Physiology and Molecular Biology of the Phenylpropanoid Metabolism,” Annual Review of Plant Physiology and Plant Molecular Biology 40:347-369 (1989)).
Activation of PAL can lead to the accumulation of lignin, suberins, and a variety of phenolic esters that increase the strength of cell walls. (Hammond-Kosack et al., “Resistance Gene-Dependent Plant Defense Responses,” The Plant Cell 8:1773-1791 (1996)). Also, PAL is necessary for the synthesis of flavonoid derivatives that function as pigments, as well as in intracellular signaling, UV protectants, phytoalexins and coumarins, and salicylic acid. PAL is also involved in the synthesis of acetosyrnigone, a wound metabolite that serves as a signal for the activation of virulence (vir)genes in Agrobacterium tumefaciens. 
PAL is usually encoded by a small gene family of 2 to 6 members. In some plants (e.g., Solanum tuberosum), 40 PAL genes can be detected (Joos et al., “Phenylalanine Ammonia Lyase in Potato (Solanum. tuberosum L.) Genomic, Complexity, Structural Comparison of Two Selected Clones and Modes Of Expression,” European Journal of Biochemistry 204:621-629 (1992)), while in loblolly pine (Pinnus taeda), there seems to be only one (Whetten et al., “Phenylalanine Ammonia Lyase from Loblolly Pine: Purification of the Enzyme and Isolation of Complementary DNA Clones,” Plant Physiology 98:380-386 (1992)). It is supposed that different members of the family respond differentially to the induction signals, either in their kinetics of induction, or accumulation of transcripts.
PAL activity is mainly regulated at the level of transcription, by the synthesis of new mRNA (Lois et al., “A Phenylalanine Ammonia-Lyase Gene from Parsley: Structure, Regulation and Identification of Elicitor and Light Responsive Cis-Acting Elements,” EMBO Journal 8:1641-1648 (1989)). Perturbation of the normal PAL expression in transgenic plants generates abnormal development phenotypes (Elkind et al., “Abnormal Plant Development and Down-Regulation of Phenylpropanoid Biosynthesis in Transgenic Tobacco Containing Heterologous Phenylalanine Ammonia-Lyase Gene,” Proceedings of the National Academy of Sciences of the USA 87:9057-9061 (1990)). Accumulation of PAL transcripts has been observed in the presence of developmental cues, wounding (Ohl et al., “Functional Properties of a Phenylalanine Ammonia-Lyase Promoter from Arabidopsis,” The Plant Cell 2:837-848 (1990)), hypersensitive response (Dong et al., “Induction of Arabidopsis Defense Genes by Virulent and Avirulent Pseudomonas Syringae Strains and by a Cloned Avirulence Gene,” The Plant Cell 3:61-72 (1991)), ozone fumigation (Sharma et al., “Ozone induced Expression of Stress Related Genes in Arabidopsis Thaliana,” Plant Physiology 105:1089-1096 (1994)), and insect saliva (Hartley et al., “Biochemical Aspects and Significance of the Rapidly Induced Accumulation of Phenolics in Birch Foliage,” Tallamy, ed., Phytochemical Induction in Herbivores, New York: John Wiley, pp. 105-132 (1991)).
Every cell in the plant has to fulfill basic needs to survive. For this purpose, a set of proteins is constantly present in the cell which is involved in functions such as membrane traffic, membrane stability, cytoplasm organization, transcription apparatus, and primary metabolic pathways. Genes encoding these proteins are called “housekeeping genes,” and they are controlled by promoters that are active almost permanently during the cell cycle. Such promoters are called “constitutive.” Since the default state of eukaryotic promoters is “off” (contrary to prokaryotic promoters; Lewin, “Genes V.,” Oxford University Press, Oxford, UK (1994)), constitutive promoters must contain structural features that enable them to remain active in several tissues and during the multiple developmental stages of the plant. By sequencing, comparing, and modifying plant promoters, it has been possible to identify functional components in their DNA sequence.
In contrast to housekeeping genes, some genes encode products that are only required under special conditions related to developmental stages of the plant, environmental stress, or pathogen attack. These genes contain “inducible promoters” that can be turned on quickly by an inducer agent and are active for a limited length of time before they are turned off again. In the absence of an inducer, the DNA sequences or genes will not be transcribed. The inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide, or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, or through the action of a pathogen or disease agent, such as a virus or fungus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating, or by exposure to the operative pathogen. In addition, inducible promoters include promoters that function in a tissue specific manner to regulate the gene of interest within selected tissues of the plant. Examples of such tissue specific promoters include seed, flower, or root specific promoters as are well known in the field (see e.g., U.S. Pat. No. 5,750,385 to Shewmaker et al.). For transgenic plants, these promoters are of interest if accumulation of the protein product, for biological or marketing reasons, is desired in certain tissues, or at certain times.
Genetic engineering provides valuable tools for studying promoter activity. By making constructs in which a reporter gene is fused under the control of a promoter sequence, it is possible to observe the specific activity of the promoter by monitoring the expression of the reporter gene (Herrera-Estrella et al., “Chimeric Genes as Dominant Selectable Markers in Plant Cells,” EMBO Journal 2:987-995 (1983)). Gene fusion not only provides a way to eliminate variables associated with post-transcriptional regulation from the experiment but also allows comparisons among different promoters or among variations of the same promoter (promoter deletion analysis).
Although a powerful tool in the study of gene control, gene fusion sometimes requires additional analyses in order to provide meaningful results. This is especially true in the characterization of tissue specific promoters. In this case, sequences containing the information for tissue specificity are not only present in the 5′ upstream region of the gene, but can also occur in the downstream coding region or even in the 3′ end region (Fu et al., “High-Level Tuber Expression and Sucrose Inducibility of a Potato Sus4 Sucrose Synthase Gene Require 5′ and 3′ Flanking Sequences and the Leader Intron,” Plant Cell 7:1387-1394 (1995); and Sieburth et al., “Molecular Dissection of the Agamous Control Region Shows that Cis Elements for Spatial Regulation are Located Intragenically,” The Plant Cell 9:355-365 (1997)).
So far, the Colombia cultivar has remained resistant to coffee rust and other coffee pathogens in Colombia. It is possible, nonetheless, that new strains of Hemileia vastatrix could be selected for virulence or that new diseases could be introduced to the hemisphere that are pathogenic to the Colombia cultivar. For these reasons, components of the Colombia variety are permanently being screened for resistance against important pathogens, and, taking advantage of the dynamic nature of a multiline, components are continually added to or removed from the final product delivered to the farmers.
Furthermore, there is no known resistance to the coffee berry borer, Hypothenemus hampei, at the cultivar or species levels, and, at the time the borer appeared in Colombia (1988), no resistant material had been introduced into the Colombia cultivar. The main obstacle to developing cultivars resistant to coffee berry borer is the lack of resistant parents. Pathogens create a constant selective pressure on the plant population, which has resulted in the co-evolution of the mechanisms of attack and defense (Agrios, “Plant Pathology, 4th Ed.,” Academic Press, San Diego, U.S.A., 635p (1997)). The natural conditions in which this process develops are altered by the agricultural practice of monoculture which has resulted in enormous crop losses caused by pathogens that have evolved while the host has been held stable.
Because of the low variability present in natural populations of C. arabica, resistance must be sought in other species. Even if a related species is found to be resistant, transfer of resistance genes from a wild relative of coffee into the cultivated varieties of C. arabica can be difficult or impossible due to interspecific incompatibility and the diploid-tetraploid nature of the cross. Despite that, by use of tissue culture for embryo rescue, crosses between species that are very distant from each other in the phylogenetic tree can be enabled; however, the long term backerossing program needed to select away from characters introgressed from the wild species makes this a difficult option.